Device for multiple simulataneous gene transfer

ABSTRACT

The present invention relates to a device for membrane passage, which comprises at least two magnetic fields generating means, each of which can generate an alternating magnetic field in a spatially limited area located in or in the immediate vicinity of the means, and a separate sample containing membrane-enveloped biological material in each spatially limited area, the device being further connected to a computer program which controls the magnetic field generating means with respect to point of time and duration for activating each individual means.

FIELD OF THE INVENTION

The present invention relates to equipment for insertion of molecularunits in membrane-enveloped biological material in multiple samples bymeans of a magnetic alternating field and a computer program, methodsfor insertion of molecular units in membrane-enveloped biologicalmaterial in multiple samples by means of a magnetic alternating fieldand a computer program and uses thereof.

BACKGROUND ART

Magnetism and magnetically susceptible particles have been used for along time in various biochemical and medical applications, ref. 1. Whenparamagnetic materials are exposed to an external alternatinghomogeneous magnetic field, heat and motion are generated. Thisgeneration of heat/motion, in particular in combination withsuperparamagnetic nanoparticles, is used, inter alia, for transfectionof cells, ref. 2. The present invention comprises a subcomponent whichis based on a device as described in ref. 2.

To allow a comparative study of various samples in one or more differentmagnetic fields where all samples must be treated in exactly the sameway, it is necessary for the transfection of all samples to be carriedout simultaneously. With the devices that are currently available, thisis not possible since each sample in the series must be mixed andtreated individually. Then, on the one hand the cell suspension has tobe incubated on ice, in which case the cell suspension cannot be assumedto be exactly the same in all samples, and on the other hand it isdifficult to treat so many samples in exactly the same way.

By means of the present invention, based on a construction comprising atleast two coils which can be supplied with current simultaneously orsequentially and which can be controlled individually by a computerprogram or software, this need is satisfied. The advantages of thepresent invention also include that it is adjusted to standardmultisample containers, so-called microtiter plates, comprising forinstance 48 or 96 wells, and that it can easily be equipped with arobotic sample handling system or be included as part of existingautomated systems where the transfection of cells constitutes part of alonger sequence of measures. A further advantage is that thetransfection can be made more efficient in terms of time since multiplesamples can be treated in parallel.

A device for increasing the thermal and/or kinetic energy ofmagnetically susceptible particles is previously known, said devicecomprising at least two magnetic field generating means, of which atleast one is a coil, between which means an alternating magneticgradient field can be generated in a spatially limited area, in whichhuman or animal tissue can be inserted, said alternating magneticgradient field causing in an increase of the thermal and/or kineticenergy of the magnetically susceptible particles which have beensupplied to said tissue, the increased thermal and/or kinetic energy ofthe magnetically susceptible particles selectively reducing,deactivating or destroying endogenic or exogenic biological structuresin said tissue, ref. 3. In the present invention, the coils are not usedto generate an alternating gradient field in a spatially limited area,but to generate homogeneous alternating magnetic fields in two or morespatially limited areas Moreover the coils are controlled by a computerprogram, individually or simultaneously.

A device based on a plurality of coils for stimulation of respiratorymuscles, ref. 4, and a device for treatment of cancer, ref. 5, arepreviously known. These devices are designed for medical therapy andtherefore cannot be used in simultaneous gene transfer in multiplesamples.

The invention can be used, not only for transfection, but also formembrane passage in different types of gene transfers and moleculetransfers (exemplified by DNA, RNA, genes, protein, peptides,antibodies, synthetic molecules and viruses) in the membrane-envelopedbiological material (exemplified by cells, cell components, liposomesand viruses). Synthetic molecules for insertion in membrane-envelopedstructures can be exemplified by fluorescent molecules and colouringmatters. A conceivable application of the invention according to theinvention thus is insertion of synthetic molecules which result in easysearch of the coloured or fluorescent biological material.

SUMMARY OF THE INVENTION

According to the present invention, a device is provided, which solvesthe problems in simultaneous treatment of multiple samples in genetransfers, such as transfection processes. Thus, a multisample handlingdevice is provided, which can generate homogeneous alternating magneticfields in two or more spatially limited areas. Each individual coil canbe controlled and checked separately by a computer program. In thespatially defined areas, samples containing suspensions are inserted,which may contain cells, viruses, plasmids, DNA, RNA and magneticallysusceptible particles. The increased thermal and/or kinetic energy ofthe magnetically susceptible particles causes transport of the molecularunits, such as DNA, RNA, protein, peptides, into the cells or virusparticles.

In one embodiment of the invention, a device for membrane passage isprovided, which contains at least two magnetic field generating means,each of which can generate an alternating magnetic field in a spatiallylimited area located in or in the immediate vicinity of said means, anda separate sample containing membrane-enveloped biological material andmagnetically susceptible particles can be inserted in each spatiallylimited area, said device being further connected to a computer programwhich controls the magnetic field generating means with respect to pointof time and duration for activating each individual means. By theexpression “activating” as used herein is thus meant the control,performed by the program, of the strength of the applied magnetic field,its frequency and of how long it is to be generated. It is a greatadvantage that each individual means can be controlled individually orsimultaneously.

In another embodiment of the invention, a method is provided forinsertion of molecular units in multiple samples containingmembrane-enveloped biological material and magnetically susceptibleparticles simultaneously or sequentially, in which

-   -   a) each sample is inserted in a spatially limited area located        in or in the vicinity of a magnetic field generating means;    -   b) the magnetic field generating means generates an alternating        magnetic field by a computer program which controls the magnetic        field generating means with respect to point of time and        duration for activating each individual means,    -   c) the molecular units are inserted in the membrane-enveloped        biological material through the pores that are produced by the        generated alternating magnetic field.

In the present invention, the molecular units are selected among DNA,RNA, genes, proteins, antibodies and peptides, and themembrane-enveloped biological material is selected among stem cells,mammalian cells, malignant cells, plant cells, nerve cells, bacteria,viruses, cellular organelles, cell-membrane-enveloped structures,cell-wall-enveloped structures, liposomes, protozoa, parasites andcombinations thereof.

In one embodiment of the invention, the device comprises at least twocoils which are supplied with two independent alternating currents ofthe same frequency and amplitude.

Moreover the device can suitably be equipped with a thermostat foraccurate control of the temperature of said samples and/or with avariable timing for accurate control of the time during which saidsamples are exposed to the alternating magnetic field.

In an embodiment of the device, the alternating magnetic field shiftswith a frequency of 1 MHz and the field strength in said coils amountsto 100 Örstedt.

Cells/Cell components/Liposomes/Viruses to be treated may consist ofbacteria, protoplasts, plant cells, mitochondria, viruses, protozoa,animal cells, mammalian cells and stem cells.

The magnetically susceptible particles suitably comprise a core of ametal oxide and an envelope containing Concanavalin A, other lectins,cell-binding proteins, cell-binding peptide sequences, antibodies orparts thereof and having a size of 0.1-300 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the construction of an embodimentof the device according to the invention for generating 16 multiplealternating magnetic fields.

FIG. 2 is an illustration of an electronic circuit that can be used tosupply the coils in one embodiment of the device according to theinvention with an alternating current.

FIG. 3 is an illustration of a connection of each individual coilelement, based on an oscillating circuit.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, a new device for use in multiplegene transfer is provided.

The invention will now be described in more detail by means of thedrawings which illustrate embodiments of the invention.

In order to generate multiple alternating magnetic fields, a deviceaccording to the invention is required, for instance as illustrated inFIG. 1. The functional principle is based on, for instance, 16 coilsmarked A-P being arranged in two juxtaposed rows. A control unit Q, bysoftware in the form of a computer program, directs the current throughthe coils so that each coil can be supplied with current individuallyand independently of the other coils. This current supply, whosefrequency and amplitude are controlled by the oscillator R, makes itpossible for the coils to generate an alternating magnetic field in apredetermined sequence. In the coils, or in the their immediate vicinity(i.e. within a distance of 5 cm), a sample container containing 16samples marked A1-P1 is arranged. Gene transfer in the samples isobtained after exposing the samples to the alternating magnetic field inthe coils.

The present invention also comprises variants in which, for instance,current intensity, current control by software, number of coils, designof the coils and temperature control may be varied.

FIG. 2 illustrates an example of an electronic circuit that may be usedto supply the coils in the device according to the invention with analternating current. The circuit comprises an oscillator 4 based on thecircuit XR2206, whose output signal 5 is amplified by a power amplifierstep 6 connected in parallel and based on 5 circuits of the type PBD3548/l (made by Ericsson), whose output signal 7 can drive analternating current (max 1 MHz, 10 A) through one or more coils.

It is obvious to a person skilled in the art that the electronic circuitdescribed above in FIG. 2 can easily be modified and that the sameresult can be obtained by various alternative prior-art connections ofoscillators and power amplifiers. Such alternative connections arewithin the scope of the present invention.

An example of the connection of the coils implies that each coilconstitutes part of an oscillating circuit consisting of a 0.5 Ωresistance, a 127 pF capacitor and a 200 μH coil, connected in series,said oscillating circuit being supplied with alternating current asshown in FIG. 3.

Each coil in FIG. 1 constitutes part of an oscillating circuit accordingto FIG. 3 which, in turn, is connected to an oscillator and a drivecircuit according FIG. 2. This means that there is a total of 16 sets ofeach component. In an alternative embodiment, 16 relays are used, whichcan independently connect the respective coils (and their oscillatingcircuit) to a single oscillator and drive circuit according to FIG. 2.The relays are controlled by software or a logically based system.

It is obvious to a person skilled in the art that the example describedabove can easily be modified and that the same result can be obtained byvarious alternative connections and coils.

EXAMPLES Example 1 Comparative Study of the Effect of DifferentCell-Binding Epitopes on Transformation of E. coli with pUC18

A colony of E. coli BL121 was grafted from a minimum media plate to 5 mlculture medium and was then placed in a shaking incubator over night.The next day 0.4 ml of the overnight culture was grafted to 40 ml of newculture medium. The culture flask was again placed in the shakingincubator and the growth rate was controlled by withdrawing samples thatwere analysed with regard to absorbance at 600 nm. At the absorbance 600nm=0.4 the cultivation was interrupted. The cells were centrifuged at5000 g for 10 min. The cell pellets were washed with 40 ml 0.05 M CaCl₂,1 mM MnCl₂, 0.15 m NaCl. The cells were centrifuged for 10 m at 5000 gand the cell pellets were resuspended in 4 ml 0.05 M CaCl₂, 1 mM MnCl₂.

The following was added to a microtiter plate with 48 sample wells:

Wells 1-8: Plasmid pUC18 (30 μg/ml) as Follows

-   well1=0 μl-   well2=0.1 μl-   well3=0.5 μl-   well4=0.75 μl-   well5=1.0 μl-   well6=1.5 μl

10 μl cell suspension was added and the samples were incubated* for 10min at room temperature in a shaking incubator, then 0.05 M CaCl₂, 1 mMMnCl₂ was added so that each sample had a final volume of 200 μl.*Incubation does not take place until all wells have been filled withthe respective samples.

Wells 7-16: 1 μl pUC18 (30 μg/ml) as distributed in wells 1-6 above, 10μl cell suspension and the samples were incubated* for 10 min at roomtemperature in a shaking incubator. Then 20 μl Con-A ferrofluid((μ_(r)=1.00200), protein concentration: 0.01 mg/ml) was added.*Incubation does not take place until all wells have been filled withthe respective samples.

0.05 M CaCl₂, 1 mM MnCl₂ was added so that each sample had a finalvolume of 200 μl.

Wells 17-24: 1 μl pUC18 (30 μg/ml) as distributed in wells 1-6 above, 10μl cell suspension and the samples were incubated* for 10 min at roomtemperature in a shaking incubator. Then 50 μl Con-A ferrofluid((μ_(r)=1.00200), protein concentration: 0.01 mg/ml) was added.*Incubation does not take place until all wells have been filled withthe respective samples.

0.05 M CaCl₂, 1 mM MnCl₂ was added so that each sample had a finalvolume of 200 μl.

Wells 25-32: 1 μl pUC18 (30 μg/ml) as distributed in wells 1-6 above, 10μl cell suspension and the samples were incubated* for 10 min at roomtemperature in a shaking incubator. Then 20 μl antiOmpA ferrofluid((μ_(r)=1.00200), protein concentration: 0.01 mg/ml) was added.*Incubation does not take place until all wells have been filled withthe respective samples.

0.05 M CaCl₂, 1 mM MnCl₂ was added so that each sample had a finalvolume of 200 μl.

Wells 33-40: 1 μl pUC18 (30 μg/ml) as distributed in wells 1-6 above, 10μl cell suspension and the samples were incubated* for 10 min at roomtemperature in a shaking incubator. Then 50 μl antiOmpA ferrofluid((μ_(r)=1.00200), protein concentration: 0.01 mg/ml) was added.*Incubation does not take place until all wells have been filled withthe respective samples.

0.05 M CaCl₂, 1 mM MnCl₂ was added so that each sample had a finalvolume of 200 μl.

Wells 41-48: 1 μl pUC18 (30 μg/ml) as distributed in wells 1-6 above, 10μl cell suspension and the samples were incubated* for 10 min at roomtemperature in a shaking incubator. Then 50 μl COO⁻ ferrofluid(μ_(r)=1.00200) was added.*Incubation does not take place until all wells have been filled withthe respective samples.

0.05 M CaCl₂, 1 mM MnCl₂ was added so that each sample had a finalvolume of 200 μl.

The microtiter plate was incubated for another 15 min at roomtemperature, after which the entire plate was put down in the devicedescribed in this application and exposed to a magnetic field 1 Mhz, 100Oe for 20 s.

200 μm culture medium was added by a pipette to each sample, and thenthe plate was arranged for incubation at 37° C. for 45 min.

Samples of 100 μl were spread on agar plates containing IPTG, ampicillinand X-gal. The plates were incubated over night at 37° C., after whichthe number of blue colonies (positive transformants) was counted foreach individual plate.

The results from this experiment were used to verify the effect of thedifferent ferrofluids on the transfection at a varying content of DNAplasmid. This study of such a large number of samples cannot be carriedout if each sample in the series were to be mixed and treatedindividually since on the one hand the cell suspension has to beincubated on ice, in which case the cell suspension cannot be assumed tobe exactly the same in all samples and, on the other hand, it isdifficult to treat so many samples in exactly the same way. Bymultisample treatment, it is further possible to obtain much more basicdata in a considerably shorter time for different transfection studies,which makes it easier to guarantee the results.

REFERENCES

-   1. Jordan A., Wust P., Scholz R., Faehling H., Krause J. & Felix R.    Magnet Fluid Hyperthermia, 569-597, in Scientific and Clinical    Applications of Magnetic Carriers, edited by Häfeli U., Schutt W.,    Teller J. and Zborowski M. Plenum Press 1997.-   2. Fredriksson S., Kriz D., Sep. 8, 1999, WO 01/18168.-   3. Fredriksson S., Kriz D., Sep. 8, 1999, WO 01/17611.-   4. Mecchlenburg D., Oct. 17, 1997, WO 99/20339.-   5. Gordon R. T., Sep. 23, 1983, U.S. Pat. No. 4,662,359.

1. A device for membrane passage comprising at least two magnetic fieldgenerating means, each of which can generate an alternating magneticfield in a spatially limited area located in or in the immediatevicinity of said means, and a separate sample containingmembrane-enveloped biological material and magnetically susceptibleparticles can be inserted into each spatially limited area, said devicebeing further connected to a computer program which controls themagnetic field generating means with respect to point of time andduration for activating each individual means.
 2. A device as claimedin. claim 1, wherein the magnetic field generating means are coils andcan consist of an electric conductor which is wound on a core consistingof air or a polymer or a substance having a relative magneticpermeability greater than
 2. 3. A device as claimed in claim 1, whereinthe magnetic field generating means are supplied with alternatingcurrents of the same frequency and amplitude, and that said means aresimultaneously supplied with said current.
 4. A device as claimed inclaim 1, wherein said device is equipped with a thermostat for accuratecontrol of the temperature of said samples and/or that it is equippedwith a variable timing for accurate control of the time during whichsaid samples are exposed to the alternating magnetic field.
 5. A deviceas claimed in claim 1, wherein the alternating magnetic field shiftswith a frequency in the range 10 kHz to 100 MHz and that the fieldstrength in said coils amounts to min 0.1 and max 1000 Örstedt.
 6. Adevice as claimed in claim 1, wherein said. magnetic field generatingmeans are arranged in the form of a matrix so that a standard microtiterplate with 48 or 96 wells can constitute a sample container.
 7. A deviceas claimed in claim 1, wherein said device comprises an automaticrobotic sample handling system.
 8. A device as claimed in claim 1,wherein the membrane-enveloped biological material is selected amongstem cells, mammalian cells, malignant cells, plant cells, nerve cells,bacteria, viruses, cellular organelles, cell-membrane-envelopedstructures, cell-wall-enveloped structures, liposomes, protozoa,parasites, and combinations thereof.
 9. A device as claimed in claim 1,wherein the magnetically susceptible particles comprise a core of metaloxide and an envelope containing Concanavalin A, lectins, cell-bindingproteins, cell-binding peptides, RNA, DNA, antibodies or genes.
 10. Amethod for insertion of molecular units in multiple samples containingmembrane-enveloped biological material and magnetically susceptibleparticles simnultaneously or sequentially, in which d) each sample isinserted in a spatially limited area located in or in the vicinity of amagnetic field generating means; e) the magnetic field generating meansgenerates an alternating magnetic field by a computer program whichcontrols the magnetic field generating means with respect to point oftime and duration for activating each individual means, and f) themolecular units are inserted in the membrane-enveloped biologicalmaterial through the pores that are produced by the generatedalternating magnetic field.
 11. A method as claimed in claim 10, whereinthe molecular units are selected among DNA, RNA, genes, proteins,antibodies, peptides and synthetic molecules.
 12. A method as claimed inclaim 10, wherein. the membrane-enveloped biological material isselected among stem cells, mammalian cells, malignant cells, plantcells, nerve cells, bacteria, viruses, cellular organelles, cellmembrane-enveloped structures, cell-wall-enveloped structures,liposomes, protozoa, parasites, and combinations thereof.
 13. A methodas claimed in claim 10, wherein the magnetically susceptible particlescomprise a core of a metal oxide and an envelope containing ConcanavalinA, lectins, cell-binding proteins, cell-binding peptides, PKA, DNA,antibodies or genes.
 14. A method for membrane passage inmembrane-enveloped structures, gene transfer or transfection comprisingutilizing the device of claim
 1. 15. A method for insertion of molecularunits in membrane-enveloped biological material selected among stemcells, mammalian cells, malignant cells, plant cells, nerve cells,bacteria, viruses, cellular organelles, cell-membrane-envelopedstructures, cell-wall-enveloped structures, liposomes, protozoa,parasites and combinations thereof, comprising utilizing the device ofclaim
 1. 16. A method claim as claimed in claim 15, wherein themolecular units are selected among DNA, RNA, genes, proteins,antibodies, peptides and synthetic molecules.
 17. A robotic samplehandling system or automatic sample preparation system for gene transfercomprising the device of claim
 1. 18. A device as claimed in claim 2,wherein the magnetic field generating means are supplied withalternating currents of the same frequency and amplitude, and that saidmeans are simultaneously supplied with said current.
 19. A device asclaimed in claim 2, wherein said device is equipped with a thermostatfor accurate control of the temperature of said samples and/or that itis equipped with a variable timing for accurate control of the timeduring which said samples are exposed to the alternating magnetic field.20. A device as claimed in claim 3, wherein said device is equipped witha thermostat for accurate control of the temperature of said samplesand/or that it is equipped with a variable timing for accurate controlof the time during which said samples are exposed to the alternatingmagnetic field.
 21. A method as claimed in claim 11, wherein themagnetically susceptible particles comprise a core of a metal oxide andan envelope containing Concanavalin A, lectins, cell-binding proteins,cell-binding peptides, PKA, DNA, antibodies or genes.
 22. A method asclaimed in claim 12, wherein the magnetically susceptible particlescomprise a core of a metal oxide and an envelope containing ConcanavalinA, lectins, cell-binding proteins, cell-binding peptides, PKA, DNA,antibodies or genes.